Fluorescence polarization and fluorescence intensity measurements provide a powerful means by which macromolecular association reactions can be studied. These fluorescent techniques have been applied to study antigen-antibody, peptide-antibody, hapten-antihapten, protein-ligand, and protein-DNA interactions.
The inherent sensitivity of fluorescence measurements can be used in monitoring the extent of reaction as a fluorescent reactant, F, combines with its macromolecular partner, R:
                              F          +          R                ⁢                              ⇌                          k                              -                1                                                          k            1                          ⁢                  F          -          R                                    Eq        .                                  ⁢                  (          1          )                    where k1 is the forward reaction and k−1 is the back reaction such that (k1)/(k−1)=K(eq).
The investigator can choose to follow changes in the fluorescence polarization UP) and/or the fluorescence intensity (FI). If the reactants do not have natural fluorescence, as in the case of many hapten- or antigen-antibody systems, one of the reactants can be covalently labeled with a fluorescent tag. An increase in the fluorescence polarization of F usually occurs during combination with R, even if there are no concomitant changes in the fluorescence intensity. This is because the polarization increase reflects a slowing down of the rotary brownian motion of the smaller ligand, F, when it becomes attached to the larger species, R. R is in many instances an antibody or a fragment of an antibody, such as an Fab or Fab2 (dimer). Equilibrium fluorescence polarization and intensity measurements can be determined in a direct readout polarometer capable of measuring both the degree of fluorescence polarization and the fluorescence intensity of a solution.
Immunoassays have been used in an effort to improve upon the success in detecting analyte substances at very low levels. For example, the use of such techniques has been prompted by the extraordinary successes that have been achieved in the measurement of biological substances by specific immunological reagents and techniques. Available evidence indicates that specific binding antibodies can be obtained even against low molecular weight organic compounds, such as pesticides or other haptens.
Any means of applying an immunochemical reaction to a detection problem ultimately relies upon a binding reaction occurring between a substance (antigen or hapten) and its specific antibody. One means by which this interaction can be employed in measurement and detection has come to be known as “competitive binding assay”. In principle, this method requires two reagents. These are a labeled form of the substance to be detected or measured, and an antibody or receptor specifically directed against the substance. The principle of the assay involves a preliminary measurement of the binding of the labeled hapten or antigen (substance being detected) with its antibody and then, a determination of the extent of the inhibition of this binding by known quantities of the unlabeled hapten or antigen, which corresponds to the unknown. From these data, a standard curve can be constructed which shows the degree of binding by the labeled hapten or antigen under certain specified conditions as a function of concentration of the unlabeled hapten or antigen or unknown added.
One way of implementing an immunoassay is to employ a fluorescent label. Usually, fluorescent labeling of one of the reagents e.g. the hapten is important in the carrying out of the immunoassay by means of fluorescence polarization and/or fluorescence intensity measurements. Unlike other immunoassays, such as ELISA, no physical separation of bound from free forms of the labeled hapten is necessary. Therefore a simple rapid optical measurement yields the essential information without physical separation of bound and free labeled materials.
Direct readout polarometers (having a machine time-constant of 0.1 seconds to several minutes) can be used to study slow kinetic reactions (reaction time-constant 10 seconds or longer) as well as reactions near or at equilibrium. These direct readout polarometers (defined as “static” polarometers) are capable of measuring both the degree of fluorescence polarization, P=(V−H)/(V+H) and the fluorescence intensity (V+H). V−H can also be measured and utilized as a parameter. Some antigen-antibody reactions can be slow enough such that they can be studied with the static polarometer. Other antigen-antibody reactions as well as many hapten-antibody reactions occur too rapidly (reaction milliseconds to seconds) to be monitored by the static fluorescence polarization or fluorescence intensity device. Fast reaction technology (e.g. stopped-flow methodology) has been combined with fluorescence polarization and fluorescence intensity techniques to study rapid hapten-antibody, rapid antigen-antibody, rapid enzyme-substrate, rapid substrate-receptor reactions. Such rate assays should lead in principle to simplified and improved assays even when applied to the analysis of real analytes. Yet currently there are few fluorescence polarization or fluorescence intensity rate immunoassays as well as other rate assays involving substrates and receptors. This is because fluorescence polarization and fluorescence intensity stopped-flow devices are expensive, somewhat complicated, and at times limited by background problems. “Static” fluorescence polarometers rate immunoassays require large dilutions of fluorescent reactants and analytes to slow down these fast reactions so that a reasonable time frame (seconds to minutes) can be attained. Others methods used to slow down the reaction are pH and/or temperature changes. These necessary reactant and analyte assay changes (pH, temperature, dilution) lead to background problems and loss of sensitivity. These background problems are severe because the background signal becomes large relative to the specific immunoassay (or assay) signal. The background signal is related to noise originating from photomultiplier noise, solution matrix light scattering and a variety of fluorescent signals coming from irrelevant non-specific binding substances. Therefore, it would be particularly advantageous to provide a competitive-type fluorescence polarization and/or fluorescence intensity immunoassay in which the rate of association reactions of the labeled substance with its specific antibody is substantially reduced. This would allow the investigator to successfully analyze real samples by fluorescence polarization or fluorescence intensity assays without the need for special instrumentation or expensive fast reaction methodology, such as stopped-flow techniques.
As for any homogeneous immunoassay, a limitation of fluorescence polarization and/or fluorescence intensity assays, when applied to analysis of real samples, has been the background signal, which is caused partly by scattered light and partly by the fluorescence of the sample matrix. Scattered light can be caused by macromolecules, such as proteins, and also by a fluorescent label that has a small Stokes shift. One approach to avoid or minimize the background signal has been to measure the rate of the immunochemical reaction between the fluorescent reactant (antigen or hapten) and its specific antibody as the analytical parameter, instead of the signal obtained when the reaction reaches or is close to the equilibrium. Although it is desirable to measure the rate of the reaction to obtain more accurate measurements, the rate of a competitive antigen-antibody or hapten-antibody reaction is usually very fast. Therefore, it has been necessary to obtain data using fluorescence polarization and or fluorescent intensity methods in combination with stopped-flow methods.
Therefore, there is a need to provide improved assays for detecting the presence and/or amount of an analyte in a sample. In particular, it would be advantageous to provide a competitive-type fluorescence polarization and/or fluorescence intensity immunoassay in which the rate of the association reaction of a labeled substance (antigen or hapten) with its specific antibody is substantially reduced. This would allow the investigator to successfully analyze real samples without the need for special instrumentation, such as the stopped-flow fluorescence polarometer, as well as increasing the performance of the stopped-flow devices themselves.